This invention relates to methods for identifying substances capable of inhibiting the enzymes dehydroquinate synthase (DHQS) and/or dehydroquinase (DQ). It further relates to DHQS and/or DQ assays for identifying activity in a sample and to test kits for identifying substances capable of inhibiting dehydroquinate synthase and/or dehydroquinase.
The shikimate pathway is an ancient pathway that is involved in primary and secondary metabolism and is found in all prokaryotes, many lower eukaryotes and plants, but not in mammalian cells. In primary metabolism the function of the pathway is to provide the precursors for the production of the aromatic amino acids and para-aminobenzoic acid. The shikimate pathway includes the enzymes and metabolites formed by converting 3-deoxy D-arabino-heptulosonic 3-phosphate (DAHP) to chorismic acid, the trifurication point for the three pathways leading to the production of tryptophane, tyrosine and phenylalanine.
In some microbial eukaryotes and prokaryotes, two of the products of the shikimate pathway (dehydroquinate and dehydroshikimate) are also shared by the quinate utilisation (qut) pathway (Hawkins et al., Molec. Gen. Genet. 214, 224-231, 1988). The qut pathway is a dispensable carbon utilisation pathway and the application of metabolic control analysis has shown that the common intermediates approximate an open pool and can be fluxed within and between the shikimate and qut pathways (Lamb et al., Molec. Gen. Genet. 227, 187-196, 1991; Lamb et al., Biochem. J. 284, 181-187, 1992 and Wheeler et al., Biochem. J. 315, 195-205, 1996). The biochemical relationships between the shikimate and quinate pathways are summarised in FIG. 1.
Overproduction of the gut pathway enzyme dehydroshikimate dehydratase in the absence of quinate causes an auxotrophic requirement for the aromatic amino acids due to flux of shikimate pathway dehyroshikimate to the gut pathway end point protocatechuic acid (Lamb et al., 1992). In Aspergillus nidulans for example it is advantageous to the growing mycelium that the qut pathway enzymes are only produced when quinate is available as a carbon source as their production in its absence would deplete flux in the essential shikimate pathway.
In A.nidulans the gut pathway is controlled by two transcription regulating proteins (designated QUTA and QUTR) that interact to ensure that the qut enzymes are only present when quinate is available (Beri et al., Nucleic Acids Res. 19, 7991-8001, 1987; Hawkins et al., Gene 110, 109-114, 1992; Hawkins et al., Gene 136, 49-54, 1993).
The importance of the shikimate pathway to cell viability is illustrated by experiments that result in the disruption of enzyme function. In plants, the shikimate pathway enzyme EPSP synthase has been targeted by a chemical inhibitor strategy that has resulted in the commercially successful broad range post-emergent herbicide called glyphosate.
In various microbial species, analysis of the shikimate pathway has been carried out genetically by the construction of mutants. When mutants of virulent prokaryotic or microbial eukaryotic species lacking enzymes at various steps in this pathway, the so-called aroxe2x88x92 mutants, are used to infect animals, their virulence is generally observed to be attenuated (Leech et al., J. Biol. Chem. 270, 25827-25836, 1995 and Gunel-Ozcan et al., Microbial Pathogen. 17, 169-174, 1997). After infection with aroxe2x88x92 mutants of S.typhimurium, mice are resistant to further challenge with the wild type strain. The probable reason for attenuation and immunological protection is that these aroxe2x88x92 mutants strains persist in the host-and replicate at a greatly reduced rate, thereby stimulating cell mediated immunity. The reason that aroxe2x88x92 mutants strains persist before being cleared is probably because they are able to derive sufficient quantities of the aromatic amino acids from the host cells to prevent immediate death. However, it is likely that their growth is limited by the availability of para-aminobenzoic acid.
Recently, the shikimate pathway has been characterised in apicomplexan parasites such as Toxoplasma gondii, Plasmodium falciparum (malaria) and Cryptosporidium parvum (Roberts et al., Nature 393, 801-805, 1998). Importantly, the growth of these parasites can be inhibited by the herbicide glyphosate, suggesting that the shikimate pathway will make a good target for the development of new anti-parasite agents.
The observations that both chemical and genetic inhibition of the shikimate pathway results in reduced cell viability has stimulated interest in the pathway as a possible target for drug therapy in acute microbial infection. It is likely that compounds which can inhibit the activity of shikimate enzymes will not cause cell death of the infecting microbe, but will result in attenuation in a manner analagous to the phenotype of shikimate pathway mutants. As antimicrobials, these compounds may be expected to induce stasis rather than cell lysis or death, allowing the infection to be cleared by the host""s immune system. Such an outcome is desirable as it will ameliorate the absolute selective pressure to select for the growth of resistant mutants which would inevitably be the case if the compounds used caused cell death. Additionally this strategy may also result in a degree of immune protection which may prevent reinfection. As efficacious compounds are unlikely to kill any infecting microorganisms, then the risks of toxic shock caused by, for example, bacterial protein and cellular debris will be minimised when treatment is administered.
According to the present invention there is provided a method for identifying an inhibitor of dehydroquinate synthase (DHQS) and/or dehydroquinase (DQ) comprising:
(i) contacting a test substance with DHQS and a substrate for DHQS and contacting the resulting reaction mixture with DQ, or contacting the test substance with DQ and a substrate for DQ; and
(ii) contacting the resulting reaction mixture with dehydroshikimate dehydratase (DHSD); and
(iii) determining whether the test substance inhibits the activity of DHQS or DQ.
The invention also provides:
a method of identifying DHQS activity in a sample, comprising:
(i) contacting the sample with a substrate for DHQS and contacting the resulting reaction mixture with DQ;
(ii) contacting the resulting reaction mixture with DHSD; and
(iii) determining whether the sample exhibits DHQS activity;
a method of identifying DQ activity in a sample, comprising:
(i) contacting the sample with a substrate for DQ;
(ii) contacting the resulting reaction mixture with DHSD; and
(iii) determining whether the sample exhibits DQ activity;
a test kit suitable for use in identifying an inhibitor of DHQS, which kit comprises DHQS, a substrate for DHQS, DQ, DHSD and a buffer; and
a test kit suitable for use in identifying an inhibitor of DQ, which kit comprises DQ, a substrate for DQ, DHSD and a buffer.
The invention thus provides flexible assays for dehydroquinate synthase (DHQS) and dehydroquinase (DQ). These assays couple the production of dehydroquinate (produced by dehydroquinate synthase) to either a type I or a type II dehydroquinase (which convert dehydroquinate to dehydroshikimate) and a dehydroshikimate dehydratase (which converts dehydroshikimate to protocatechuic acid). The product dehydroshikimate can be monitored at 237 nm, and/or the product protocatechuate can be monitored at either 290 nM or, after reaction with iron, at 547 nM. This means that the activity of the enzyme DHQS or DQ can be measured continuously at two different points in the uv spectrum or by a discontinuous assay in the visible spectrum.
The assay for DHQS and DQ can be used to identify inhibitors of DHQS and DQ. The fact that the assay for inhibitors can be carried out as a discontinuous assay in the visible spectrum has the advantage that cheap plastic microtitre plates can be used to detect the effects of specific compounds on DHQS and/or DQ activity. Also, the screen for inhibitor substances is not limited by the absorbance spectrum of the substance being tested because the activity of DHQS and DQ can be measured at different points in the uv and/or visible spectra. The assay is suitable for adaptation to 96 well and 384 well plate technologies and can be automated using liquid handling robots, allowing modern high throughput screening techniques to be applied. The invention therefore permits high through-put, flexible and inexpensive screening for substances which inhibit DHQS and DQ. This will increase the likelihood of potent bioavailable drugs being identified.